Augmented R-loop is a Unifying Mechanism for Myelodysplastic Syndromes Induced by High Risk Splicing Factor Mutations

Mutations in several general pre-mRNA splicing factors have been linked to Myelodysplastic Syndromes (MDS) and solid tumors. These mutations have generally been assumed to cause disease by resultant splicing defects, but different mutations appear to induce distinct splicing defects, raising a possibility for an alternative common mechanism involved. Here, we report a chain of events triggered by multiple splicing factor mutations, especially high-risk alleles in SRSF2 and U2AF1, including elevated R-loops, replication stress, and activation of the ATR-Chk1 pathway. We further demonstrate that enhanced R-loops, opposite to the expectation from gained RNA binding with mutant SRSF2, result from impaired transcription pause-release because the mutant protein lost its ability to extract the RNAPII CTD kinase P-TEFb from the 7SK complex. Enhanced R-loops are linked to compromised proliferation of bone marrow-derived blood progenitors, which can be partially rescued by RNase H overexpression, suggesting a direct contribution of augmented R-loops to MDS phenotype. Overall design: To profile the R-loop dyanmics for comparison between cells with and without expression of mutant splicing factors (SRSF2 and U2AF1) identified in patients with myelodysplastic syndromes (MDS), We performed R-ChIP experiments, a new RNaseH1-based technique recently developped in the lab to specifically capture R-loop in vivo. We established an inducible cell system to control the expression of wild-type (SRSF2 or U2AF1) or mutant [SRSF2(P95H), U2AF1(S34F), U2AF1(Q157P)] splicing factors by Dox treatment in HEK293T cells expressing mutant RNASEH1(D210N) for R-loop capture by R-ChIP. R-ChIP experiments were done for cells under two Dox treatment conditions (0 hr and 5 hr). Standard GRO-seq experiments to monitor the nascent transcription activity in wild-type and mutant cells were performed to illucidate the correlation of transcription activity with R-loop dynamics. All sequencing data were obtained from experiemnts in duplicates.